Our research efforts are directed toward investigating the life cycle and basic functions of each type of pulmonary epithelial cells in a well-defined in vitro situation. A major objective of the research is to develop conditions for the long-term cultivation of normal, differentiated epithelial cells. Such a culture offers a model system to investigate the mechanism of cell differentiation, as well as the repair of injury and the pathological response of cells to environmental toxic substances. The success in culturing normal epithelial cells from the respiratory tract is very limited, since most epithelial cells in the conventional culture condition are short lived and cellular basic functions are rapidly lost. Our approach is on one hand to eliminate those unnecessary factors from the conventional culture condition and on the other hand, to provide only required elements for cells. The first study of these series is to replace serum by the needed hormones and growth factors. Our results indicate that tracheal epithelial cells can be continuously maintained in a hormone-supplemented medium. These hormones and factors needed for tracheal cell cultures are insulin, epidermal growth factor and transferrin.